This invention is directed to methods of linking genetic information with proteins encoded by the genetic information, to plasmids that can be used in the process of covalently linking nucleic acid segments to protein encoded by the nucleic acid, and in methods for selection and screening of mutagenized genes and proteins.
The selection, identification, and evolution of in vitro modified biological receptors presents a significant experimental problem. In the broadest sense, this problem can be described as the linking or association of the genotype to its corresponding phenotype. This is a particular problem when large numbers of mutants are generated in a random or quasi-random process, such as in a combinatorial system.
A key step, therefore, in all combinatorial selection systems is the linkage of genetic information with its protein offspring. Phage display accomplishes this linkage by engineering the phage to display a small peptide on their coats. The phage contains the DNA encoding the display peptide as a part of their single-stranded viral DNA (G. P. Smith, Science 228:1315 (1985); C. F. Barbas, Curr. Opin. Biotechnol. 4:526 (1993)). Phage display is limited by the requirement that the displayed protein or peptide exist in an active conformation while covalently linked at its amino-terminus to phage coat protein. Additionally, the system is limited because proteins or peptides that would disrupt the structure of the coat protein would not be detected in this system. Phage display is also limiting because it requires the displayed protein to be active subsequent to secretion through the bacterial membrane. The displayed protein must be competent for secretion. In addition, the displayed protein must be biologically active in the macromolecular context of the phage coat.
A second approach to the problem of linking genotype to phenotype has been suggested by the peptide-on-plasmid technology (M. G. Cull et al., "Screening for Receptor Ligands Using Large Libraries of Peptides Linked to the C Terminus of the lac Repressor," Proc. Natl. Acad. Sci. USA 89:1865-1869 (1992); P. J. Schatz, "Use of Peptide Libraries to Map the Substrate Specificity of a Peptide-Modifying Enzyme: A 13 Residue Consensus Peptide Specifies Biotinylation in Escherichia coli," Bio/Technology 11:1138-1143 (1993); U.S. Pat. No. 5,270,170 to Schatz et al., issued Dec. 14, 1993). In the peptide-on-plasmid system, random peptide sequences are cloned onto the carboxyl terminus of the lac repressor. The DNA binding activity of the lac repressor is used to link the peptides to the plasmids encoding them by binding to Lac operator sequences on the plasmids. Combinatorial libraries of peptides-on-plasmids are prepared from up to 10.sup.8 E. coli transformants, which are harvested in-batch, gently lysed, and screened by conventional ligand affinity procedures, a process known as "panning." For the panning process to succeed, the fusion protein must remain connected to the plasmid. However, the half-life of the complex is only approximately 30 minutes, due to the non-covalent DNA-protein complex thus formed. In the normal panning methodology, sufficient time elapses to allow dissociation and re-association with an incorrect partner. In addition, lac binds to DNA as a tetramer, thus allowing mixed tetramers to form during and after the panning process. Thus, the linkage of one gene to its protein offspring is not insured and errors can be introduced.
As indicated above, the need for such techniques has grown as technologies for developing large libraries of nucleic acids and peptides have been developed. In particular, these libraries have proven useful for the isolation of ligands that bind biological receptors. The isolation of such ligands is critical in understanding signal transduction and in discovering new therapeutic substances and new therapeutic applications of previously known substances. In particular, the ability to synthesize DNA chemically has made possible the construction of extremely large collections of nucleic acid and peptide sequences as potential ligands. Recently developed methods allow efficient screening of libraries for desired binding activities (Pluckthun & Ge, Angew. Chem. Int'l. Ed. Enql. 30:296-298 (1991)). Other techniques, such as nucleic acid amplification techniques, particularly polymerase chain reaction (PCR) methodology, have also contributed to this area. For example, RNA molecules with the ability to bind a particular protein (Tuerk & Gold, Science 249:505-510 (1990)) or a dye (Ellington & Szostak, Nature 34:818-822 (1990)) have been selected by alternate rounds of affinity selection and PCR amplification. A similar technique was used to determine the DNA sequences that bind to human transcription factor (Thiesen & Bach, Nucl. Acids Res. 18:3203-3209 (1990)).
The power of nucleic acid synthesis, screening and amplification techniques such as PCR and solid-phase nucleic acid synthesis, greatly increases the need for an efficient and reliable method of linking nucleic acid segments to the proteins or peptides which they encode. There is also a need for plasmids that are particularly suitable for use in such linking procedures. These linking procedures and the plasmids for use with them preferably are adaptable to screening and selection techniques, as well as amplification techniques such as PCR, to allow multiple rounds of mutagenesis and selection, to study in vitro evolution of proteins and peptides. The improved methods and plasmids for such linkage are preferably also usable with a wide variety of genes and do not depend on the particular properties of the gene, protein or peptide being expressed.